雌激素受体α甲基化与肾虚型绝经后骨质疏松症关系及补肾中药的干预研究

发布时间：2018-03-06 17:31

本文选题：绝经后骨质疏松　切入点：雌激素受体α　出处：《广州中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探究骨组织中雌激素受体α(ERα)基因启动子甲基化与肾虚型绝经后骨质疏松症(PMOP)的关系以及补肾中药干预对成骨细胞内ERα基因启动子甲基化、细胞增殖及分化的影响。方法:(1)收集行髋、膝手术的PMOP女性患者20例作为观察组,行髓、膝手术的非PMOP女性患者20例作为对照组。通过问卷调查和体检的方法收集患者的一般资料和体格资料,应用双能X线骨密度分析仪检测患者腰椎2-4骨密度,应用甲基化PCR(MSP)检测患者骨组织中ERα基因启动子甲基化情况。统计学分析观察组与对照组患者骨密度、骨组织中ERα基因启动子甲基化差异及骨密度与ERα基因启动子甲基化程度的相关性。(2)二次酶消化法分离培养大鼠原代成骨细胞,用六味地黄丸和金匮肾气丸分别以10、50、100ng/μL的浓度干预处理。MSP法检测各组细胞内ERα基因启动子甲基化状况,RT-PCR与WB法检测补肾中药干预后ERαmRNA和蛋白表达变化,CCK8试剂盒检测细胞增殖活性,ALP试剂盒检测碱性磷酸酶(ALP)表达,细胞免疫组织化学染色法检测成骨细胞标志物胶原I和骨保护素的表达变化,WB法检测影响成骨细胞增殖分化的信号通路关键分子AMPK和β-catenin的表达变化。结果:(1)与正常对照组患者相比,观察组患者骨密度值降低、骨组织内ERα基因启动子甲基化增加,统计学分析表明,患者骨密度与ERα基因启动子甲基化程度呈现负相关性。(2)补肾中药干预能够抑制成骨细胞内ERα基因启动子甲基化并增加ERαmRNA和蛋白表达,其中,金匮肾气丸的作用更为明显。应用CCK8试剂盒检测细胞增殖结果显示:六味地黄丸干预后,成骨细胞的增殖活性没有明显变化;金匮肾气丸以50、100 ng/μL的浓度干预处理48 h时,能够显著地增加细胞增殖活性。同时,细胞组化检测结果显示:对于成骨细胞早期分化标志物ALP与胶原I的表达,六味地黄丸以100 ng/μL浓度干预时能够增加其表达,金匮肾气丸以50 ng/μL和100 ng/μL浓度干预处理时能够显著增加其表达,且金匮肾气丸促成骨细胞早期分化效果更为明显。此外,WB检测补肾中药干预对影响成骨细胞增殖分化的信号通路关键分子表达变化的结果显示金匮肾气丸干预处理能够降低成骨细胞内p-AMPK蛋白的表达,增加Wnt通路中β-catenin蛋白的表达。结论:患者骨密度值与骨组织内ERα基因启动子甲基化程度呈负相关;补肾中药金匮肾气丸能够显著抑制ERα基因启动子甲基化及增加ERαmRNA和蛋白表达,并通过此途径所介导的AMPK和β-catenin的作用促进成骨细胞的增殖分化。
[Abstract]:Objective: to investigate the relationship between the methylation of estrogen receptor 伪 er 伪) gene promoter and PMOP in postmenopausal osteoporosis of kidney deficiency type and the methylation of ER 伪 promoter in osteoblasts after intervention of Chinese herbs for tonifying the kidney. Methods 20 female PMOP patients undergoing hip and knee surgery were collected as the observation group. Twenty non-#en0# female patients undergoing knee surgery were used as the control group. The general and physical data of the patients were collected by questionnaire and physical examination. The 2-4 bone mineral density of lumbar vertebrae was measured by dual energy X-ray absorptiometry. The methylation of ER 伪 gene promoter in bone tissue was detected by methylated PCRG MSP.The bone mineral density (BMD) of patients in observation group and control group was analyzed statistically. The methylation difference of ER 伪 gene promoter and the correlation between bone mineral density and methylation degree of ER 伪 gene promoter in bone tissue. Using Liuwei Dihuang Pill and Jinkui Shenqi Pill to detect the methylation status of ER 伪 Gene Promoter in the cells of each Group by 100 ng / 渭 L concentration of 100.50g / 渭 L; RT-PCR and WB methods to detect the changes of ER 伪 mRNA and protein expression after intervention of Kidney tonifying herbs. The expression of alkaline phosphatase (ALP) was detected by ALP kit. Expression of osteoblast marker collagen I and osteoprotegerin were detected by immunohistochemical staining and the expression of AMPK and 尾 -catenin, the key molecules of signal pathway affecting osteoblast proliferation and differentiation, were detected by WB method. Compared with the patients in the normal control group, In the observation group, the bone mineral density decreased and the promoter methylation of ER 伪 gene increased in bone tissue. There was a negative correlation between bone mineral density (BMD) and methylation degree of promoter of ER 伪 gene. (2) intervention of traditional Chinese medicine for tonifying kidney could inhibit methylation of promoter of ER 伪 gene and increase expression of ER 伪 mRNA and protein in osteoblasts. The effect of Jingui Shenqi Pill was more obvious. The results of CCK8 assay showed that the proliferation activity of osteoblasts did not change after intervention of Liuwei Dihuang pills, and the proliferation activity of Jingui Shenqi Pill was treated with 50,100 ng/ 渭 L for 48 h. At the same time, the expression of ALP and collagen I in the early differentiation of osteoblasts could be increased by the intervention of Liuwei Dihuang pills with 100 ng/ 渭 L concentration, and the results of cell histochemical analysis showed that the expression of ALP and collagen I in the early differentiation of osteoblasts could be increased when the concentration of Liuwei Dihuang pills was 100 渭 L. Jingui Shenqi Pill could significantly increase its expression when it was treated with 50 ng/ 渭 L and 100 ng/ 渭 L concentration. Moreover, the effect of Jinkui Shenqi pills on the early differentiation of osteoblasts was more obvious. In addition, the effect of intervention of Chinese medicine for tonifying the kidney on the expression of key molecules of signal pathway affecting the proliferation and differentiation of osteoblasts was more obvious. Li can reduce the expression of p-AMPK protein in osteoblasts. Conclusion: bone mineral density is negatively correlated with the degree of methylation of ER 伪 gene promoter in bone tissue, Jinkui Shenqi Pill can significantly inhibit the methylation of ER 伪 gene promoter and increase the expression of ER 伪 mRNA and protein in bone tissue, and Jingui Shenqi Pill can significantly inhibit the methylation of the promoter of ER 伪 gene and increase the expression of ER 伪 mRNA and protein. The effects of AMPK and 尾-catenin mediated by this pathway promote the proliferation and differentiation of osteoblasts.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R259

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